The Role of HIF-1α in Sarcoma Metastasis and Response to Radiation Therapy by Minsi Zhang Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ David Kirsch, Supervisor ___________________________ Mark Dewhirst ___________________________ Christopher Kontos ___________________________ Kenneth Poss ___________________________ Jeffrey Rathmell Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2015 ABSTRACT The Role of HIF-1α in Sarcoma Metastasis and Response to Radiation Therapy by Minsi Zhang Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ David Kirsch, Supervisor ___________________________ Mark Dewhirst ___________________________ Christopher Kontos ___________________________ Kenneth Poss ___________________________ Jeffrey Rathmell An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2015 Copyright by Minsi Zhang 2015 Abstract The degree of intratumoral hypoxia is clinically correlated to poor response to therapy and increased incidence of distal spread in various cancer subtypes. Specifically, the transcription factor Hypoxia Inducible Factor-1α (HIF- 1α), which is accumulated in cells in response to a hypoxic microenvironment, is implicated in poor disease outcome associated with intratumoral hypoxia. Using novel genetically engineered mouse models of primary soft tissue sarcoma, I show that in vivo genetic deletion of HIF-1α specifically in tumor cells 1) decreases the incidence of lung metastases by limiting sarcoma collagen deposition, and 2) improves sarcoma response to radiation therapy by limiting the inflammatory response and metabolic adaptations. These results define HIF- 1α as a potential target for cancer therapy. iv Dedication This thesis is dedicated to my parents, Xianping and Weiwei, and my husband Ben for their enduring support. v Contents Abstract ................................................................................................................iv Dedication ............................................................................................................ v List of Tables ........................................................................................................ x List of Figures .......................................................................................................xi Acknowledgements ............................................................................................ xiii 1. Introduction ....................................................................................................... 1 1.1 Intratumoral Hypoxia and Hypoxia Inducible Factor-1 ............................... 1 1.2 Hypoxia, HIF-1α, and Metastasis .............................................................. 3 1.3 Hypoxia, HIF-1α, and Radiation Resistance ............................................. 4 1.4 Glucose Metabolism, Hypoxia, and the Warburg Effect ............................ 6 1.5 Inflammation, Hypoxia, and Cancer Treatment Resistance .................... 10 1.5.1 NF-κB and IRFs .................................................................................. 10 1.5.2 Tumor Inflammation and Its Association with Hypoxia and Cancer Treatment Resistance ................................................................................. 13 1.6 Primary Mouse Models of Soft Tissue Sarcoma ..................................... 14 1.6.1 Soft Tissue Sarcoma: Rhabdomyosarcoma and Undifferentiated Pleomorphic Sarcoma ................................................................................. 14 1.6.2 Site-Specific Recombination with the Cre-loxP and the Flp-FRT Systems ....................................................................................................... 15 1.6.3 Primary Mouse Models of RMS and UPS ........................................... 19 vi 1.7 Study Objectives ..................................................................................... 20 2. Materials and Methods ................................................................................... 21 2.1 in vivo Experiments ................................................................................. 21 2.1.1 Generation of Novel Mouse Models ................................................... 21 2.1.1.1 Adeno-Cre mediated sarcomagenesis ......................................... 22 2.1.1.2 4-hydroxytamoxifen mediated sarcomagenesis ........................... 23 2.1.1.3 Tamoxifen mediated Cre-ERT2 recombination of loxP sites ......... 23 2.1.2 Limb Amputation Survival Surgery ..................................................... 23 2.1.3 Image-Guided Fractionated Radiation Therapy .................................. 24 2.1.4 Injection of EF5 and Hoechst 33342 ................................................... 25 2.2 in vitro and Molecular Experiments ......................................................... 25 2.2.1 Tumor Cell Isolation and Culture ........................................................ 25 2.2.2 PCR, qRT-PCR .................................................................................. 26 2.2.3 Western Blot, Immunofluorescence, Immunohistochemistry, Fluorescence-Activated Cell Sorting ............................................................ 29 2.2.4 Tissue Microarrays ............................................................................. 31 2.2.5 RNA-Sequencing ................................................................................ 32 2.2.6 Targeted Metabolite Analysis ............................................................. 32 2.2.7 Seahorse Analysis .............................................................................. 33 2.2.8 DNA End-Joining Repair Assay .......................................................... 33 vii 3. HIF-1α Promotes Undifferentiated Pleomorphic Sarcoma Metastasis through Transcriptional Upregulation of Lysyl Hydroxylase 2 (PLOD2) ........................... 35 3.1 Introduction ............................................................................................. 35 3.2 Results .................................................................................................... 36 3.2.1 Characterization of primary mouse UPS with tumor-specific HIF-1α deletion ........................................................................................................ 36 3.2.2 Tumor-specific HIF-1α deletion in primary mouse UPS decreases metastasis in vivo ........................................................................................ 43 3.2.3 HIF-1α mediates metastasis in UPS by PLOD2 ................................. 46 3.3 Discussion ............................................................................................... 53 4. The Generation of Novel Mouse Models ........................................................ 57 4.1 Introduction ............................................................................................. 57 4.2 Results .................................................................................................... 60 4.2.1 The generation of Flp-frt regulated Cre-ERT2 alleles .......................... 60 4.2.2 The generation of a novel mouse model of soft tissue sarcoma ......... 70 4.3 Discussion ............................................................................................... 75 5. HIF-1α Promotes Soft Tissue Sarcoma Resistance to Radiation Therapy via Activation of the Glycolytic and Pro-Inflammatory Pathways .............................. 78 5.1 Introduction ............................................................................................. 78 5.2 Results .................................................................................................... 80 5.2.1 Human soft tissue sarcoma upregulates HIF-1α after chemo-radiation therapy ........................................................................................................ 80 viii 5.2.2 Deletion of HIF-1α sensitizes sarcoma cells to radiation in a cell- autonomous manner .................................................................................... 81 5.2.3 Deletion of HIF-1α does not affect DNA damage repair, cellular ROS response or autophagy following irradiation ................................................ 88 5.2.4 Deletion of HIF-1α leads to changes in mitochondrial homeostasis following irradiation ...................................................................................... 93 5.2.5 HIF-1α regulates distinct transcriptional targets after hypoxia and radiation ....................................................................................................... 97 5.3 Discussion ............................................................................................. 100 6. Conclusion .................................................................................................... 105 Appendix A: Genes differentially expressed between P7NP and P7NPH1 cells under normoxia without irradiation .................................................................... 116 Appendix B: Genes differentially expressed between P7NP and P7NPH1 cells 24 hours after 6 Gy irradiation